1. Field of the Invention
This invention relates to a general process for the preparation of single stranded cyclic oligonucleotides, DNA or RNA, as well as to new intermediates that are necessary to accomplish this process.
2. Description of Prior Art
Cyclic structures of polynucleotides (nucleic acids) are widespread in nature, forming part of the genetic material of many viruses and bacteria. There are single stranded cyclic forms of DNA (viruses) and double stranded cyclic forms of DNA (bacteria and viruses); but only single stranded cyclic structures of RNA are known (viruses).
Although some cyclic oligonucleotides of natural origin are known, most of them are of synthetic origin. The synthesis or preparation of such products has a great interest nowadays for numerous reasons, among which the following can be mentioned: cyclic oligonucleotides are good models to undertake the conformational study of nucleic acids; some of them act as inhibitors of RNA polymerase or as activators of cellulose synthetase; and, most important, single stranded DNA cyclic oligonucleotides with two pyrimidine sequences can hybridize with linear oligonucleotides (DNA or RNA) formed by polypurines, by forming triple helices with high affinity and selectivity, which represents a remarkable ability of molecular recognition that has important uses in the regulation of several biological processes (DNA replication, RNA transcription, RNA processing, translation into proteins, etc.). Promising possibilities of use as antisense oligonucleotides in genetic therapy are derived from the latter reason (for instance, use against different cancers or viral diseases such as AIDS or herpes simplex). Also promising are the possibilities of being used as probes in the diagnosis of numerous diseases, and as markers to locate specific sequences in a chromosome or in other DNA or RNA molecules. Cyclic oligonucleotides can also be used for the site specific targeting of drugs directed to a sequence or fragment of nucleic acid. For all the above-mentioned reasons there is nowadays a great interest in the preparation of cyclic oligonucleotides (see, for instance, patent WO 92/19732).
Until the mid eighties, no specific preparation processes of cyclic oligonucleotides had been described, despite the fact that Khorana et al. isolated the first of such compounds as a by-product in 1958.
At the beginning, all preparation processes involved the phosphate triester method in solution, both for obtaining the linear precursor, and for the cyclization of oligonucleotides with up to 8 nucleobases (see, for instance, M. V. Rao and C. B. Reese, Nucleic Acids Res. 1989, vol. 17, pp. 8221-39, and references cited therein). These processes are laborious and time consuming, as a consequence of working in solution. Besides, they have the main drawback of forming all the phosphate diester linkages by using a method that is slow and somewhat ineffective, especially when compared with the phosphite triester method that is routinely used nowadays for the preparation of DNA and RNA fragments.
A preparation process of cyclic oligonucleotides has been described that takes advantage of the triple helix forming ability of some cyclic oligonucleotides, in order to achieve the chemical cyclization of fully deprotected linear oligonucleotides. This process uses a linear template of polypurines that assists the approach of the ends of the oligomer, thus favoring the intramolecular reaction. But this process is limited to the preparation of cyclic sequences that contain two polypyrimidine sequences which allow the formation of the triple helix with the polypurine of the central template. Moreover, another drawback of this process is that it seems to be restricted to the preparation of rather large cyclic molecules. Thus, this process has been used for the preparation of cyclic oligonucleotides from 24 to 46 mer (base residues); but the authors themselves admit that they have failed in obtaining a 16 mer one (cf. G. Prakash and E. T. Kool, J. Am. Chem. Soc. 1992, vol. 114, pp. 3523-7; WO 92/17484).
In other cases, chemical cyclization of fully deprotected oligonucleotides has been achieved by using complementary oligonucleotide templates which form a double helix with the end fragments of the structure to be cyclized (cf. N. Dolinnaya et al., Nucleic Acids Res. 1993, vol. 21, pp. 5403-7).
In some instances the solid phase synthesis methodology has been used for the preparation of cyclic oligonucleotides, although with poor results. Thus, this methodology has been used by anchoring the oligonucleotide to the polymer through the exocyclic amino group of cytosine, and using insoluble solid supports of polyacrylamide and polyethyleneglycol-polystyrene (PEG-PS) copolymers, as well as a soluble polymeric support of polyethyleneglycol. Even though in the first preparations described the phosphate triester method was used, very recently the phosphite triester method has also been used, with PEG-PS solid supports (cf. M. R. Conte et al., Nucleosides and Nucleotides 1993, vol. 12, pp. 351-8 (1993); L. De Napoli et al., J. Chem. Soc. Perkin Trans. 1993, pp. 747-9). Nevertheless, one of the main limitations of this process is that the sequence of the cyclic oligonucleotide must contain at least one cytosine (C), since attempts to anchor adenine (A) and guanine (G) to the solid support through their exocyclic amino groups have failed.
In summary, all preparation processes of cyclic oligonucleotides known in the art have limitations or drawbacks that make them of no general use. That is, they are unsuitable to prepare any cyclic oligonucleotide sequence, of any size (until now, medium size cycles have been shown to be the most inaccessible ones).
Furthermore, the processes known in the art make use of chemical methods that are not commonly employed for the elongation of the oligonucleotide chain, for the protection of the bases or for the cyclization reaction, thus preventing the use of the large amount of knowledge related to the preparation of linear oligonucleotides, as well as to the automation of this preparation. Thus, the problem of having a general methodology for the preparation of cyclic oligonucleotides is not satisfactorily solved yet.